Lytic peptide prodrugs

ABSTRACT

A cytotoxin can be rendered non-toxic by charge neutralizing the amino acids salient to pore assembly and/or sterically inhibiting formation of the peptide&#39;s active conformation. In the presence of specific proteases, the inactive peptide or procytotoxin can be activated to assemble into its lytic conformation and selectively destroy a target cell.

This application is a continuation-in-part of application Ser. No.09/851,422 filed May 9, 2001 now U.S. Pat. No. 7,094,750.

FIELD OF INVENTION

This invention relates to methods and compositions for selectivelydestroying a target cell. Specifically, the invention relates to methodsfor making and using a procytotoxin that can be converted to a cytotoxinin a tumor-cell specific manner, such that a tumor cell is destroyed bythe activated cytotoxin.

BACKGROUND OF THE INVENTION

Present methods for tumor treatment, especially cancer treatment, remainsub-optimal and often are accompanied by severe complications. In fact,virtually all of the known therapies have serious adverse side effects,most often caused by the lack of specificity and thoroughness in thedestruction or removal of tumor or cancer cells.

For example, surgery, a common procedure for removing cancerous cellsfrom a patient, often results in incomplete removal and disfigures thepatient or interferes with normal bodily functions. Similarly,chemotherapy and radiation treatment often indiscriminately destroysnormal cells, causing unwanted side-effects, while leaving many cancercells unaffected. Chemotherapeutic agents, especially antimetabolites,while effective to varying degrees against cancer cells that arecontinuously undergoing or preparing for mitosis, are not effectiveagainst cancer cells that are in the resting (G₀) stage.

Cancer treatment is most effective when cancer cells can be eliminatedas completely as possible from the patient's body. To achieve this goal,continuous or consecutive dosages are administered to the patient.Because most available chemotherapeutic agents are also toxic to normalcells, the dose of cytotoxic drug is adjusted to the limits of toleranceto achieve the maximum destruction of malignant cells, and the intervalbetween doses must be such that the rate of tumor re-growth does notexceed tumor killing. Accordingly, in order to achieve increasedefficiency with reduced side effects, the chemotherapeutic agents shouldhave high target-cell specificity and high target-cell toxicity orpotency.

Prostate cancer is the most common form of cancer among males, with anestimated incidence of 30% in men over the age of 50. Overwhelmingclinical evidence shows that human prostate cancer has the propensity tometastasize to bone, and the disease appears to progress inevitably fromandrogen-dependent to androgen-refractory status, leading to increasedpatient mortality. This prevalent disease is currently the secondleading cause of cancer death among men. Prostate cancer metastasis isestimated to claim the lives of over 30,000 Americans each year. Inspite of considerable research into therapies for cancer, currentlyavailable treatment methods are ineffective in a significant percentageof cases.

Accordingly, there is a need for improved cancer treatment and moremethods that are not dependent upon the cell cycle of the cancer cell.Particularly, there is a need for improved treatment methods forprostate cancer. The present invention fulfills these needs and furtherprovides other related advantages.

SUMMARY OF THE INVENTION

It therefore is an object of the present invention to overcome some orall of the aforementioned deficiencies in conventional therapies,especially with regard to treating prostate cancer. To this end,compositions and methods for making a procytotoxin are described. Aprocytotoxin is provided which typically is made up of a cytotoxicpeptide bound to an inactivator via a peptide bond, and the peptide bondis susceptible to cleavage by a targeting specific protease. Thecytotoxic peptide is preferably a pore-forming cytolytic peptide. Theprocytotoxin of the present invention may also have a targeting moleculelinked to the N- and/or C-terminus of the cytotoxic peptide.

The procytotoxin of the instant invention may also have at least onelysine residue. In general, at least one lysine of the cytotoxin isbound through its ε-amino group, via an ε-γ peptide bond, to theγ-carboxyl group of a glutamic acid residue. In different embodiments,one, two, or more additional glutamate residues are bound to the oneattached to the lysine.

Generally preferred cytotoxins include Ae I, cytolysin of sea anemone,aerolysin, amatoxin, amoebapore, amoebapore homolog from Entamoebadispar, brevinin-1E, brevinin-2E, barbatolysin, cytolysin ofEnterococcus faecalis, delta hemolysin, diphtheria toxin, E1 Torcytolysin of Vibrio cholerae, equinatoxin, enterotoxin of Aeromonashydrophila, esculentin, granulysin, haemolysin of Vibrioparahaemolyticus, intermedilysin of Streptococcus intermedius, thelentivirus lytic peptide, leukotoxin of Actinobacillusactinomycetemcomitans, magainin, melittin, membrane-associatedlymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1,neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphinfragment 4, NK-lysin, paradaxin, perforin, perfringolysin O,theta-toxin, of Clostridium perfringens, phallolysin, phallotoxin,streptolysin, D,L-α-amino acid cyclic peptides, and analogs andderivatives thereof.

Particularly preferred procytotoxins have the following structures: (1)GIy-lIe-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-GIy-Leu-Pro-Ala-Leu-Ile-Ser-Trp-IIe-Lys-Arg-Lys-Arg-Gln-[Gln-Gly-Ala-Ile-Gly-Gln-Pro]-(X)(SEQ ID NOS 1 & 2, respectively), and (2)Gly-Ile-Gly-Ala-.Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Ser-Ser-Phe(or Tyr)-Tyr-Ser-Gly(or Ser)]-(X) (SEQ ID NOS 3 & 4,respectively), wherein (X) is the inactivator as described herein andthe peptide marked in brackets can be oriented in either direction. Theinactivator, for example, can be a microbead, amino acid, peptide,phage, or phage filament. Preferably, the procytotoxin further containsa targeting molecule. Still preferred, the targeting molecule is aneovascular targeting sequence of an anti-fibronectin ED-B antibody.Also preferred, the targeting molecule is an RGD targeting sequence.

Other preferred procytotoxins further are charge neutralized, inaddition to steric determinants. For example,Gly-Ile-GIy-Ala-Vat-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys(R)-Arg-Lys(R)-Arg-Gln-[Gln-Gly-Ala-Ile-Gly-Gln-Pro]-(X)(SEQ ID NOS 21 & 22, respectively), wherein (X) is the inactivator asdescribed herein, the peptide marked in brackets can be oriented ineither direction, and wherein R is independently selected from the groupconsisting of the [unmodified ε-amino group of the adjacent lysineresidue], [ε-γ]-Glu, [ε-γ]-Glu-[α-γ]-(Glu)₁₋₃, [ε-α]-(Phe)₁₋₃,[ε-α]-(Tyr)₁₋₃, [ε-α]-(Trp)₁₋₃, [ε-α]-(Lys)₁₋₃ and [ε-α]-(Arg)₁₋₃,wherein [ε-γ]represents a peptide bond between the epsilon amino groupof lysine and the gamma carboxyl group of the adjacent glutamate, [α-γ]represents a peptide bond between the alpha amino group of the firstglutamate and the gamma carboxyl group of the second glutamate, [ε-α]represents a peptide bond between the epsilon amino acid of lysine andthe alpha carboxyl group of the indicated amino acid and the subscriptindicates that additional numbers of the designated amino acid can belinked to the first via conventional peptide bonds.

Also provided, in achieving this objective of the invention, arepharmaceutical compositions that, in general, contain an inventiveprocytotoxin and a pharmaceutically suitable excipient.

Further, methodology is provided for destroying a target cellselectively. This approach typically entails contacting the target cellwith a procytotoxin, which has a cytotoxic peptide bound via a peptidebond to an inactivator, wherein the peptide bond is susceptible tocleavage by a targeting specific protease. Preferred target cells arecancer cells.

Further to the same object, a method for treating a cancer patient isprovided. In general, the method comprises administering to a patient inneed, a therapeutically effective amount of a pharmaceutical compositioncontaining procytotoxin according to the invention. Preferredcompositions contain procytotoxins based on melittin or a cytolyticpeptide derived therefrom.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that human prostate cancer cells (LNCaP) are completelylysed after 15 minutes in 50 μM amoebapore helix 3-γ-Glu-γ-Glu, whilemouse L-cells remain unaffected after 24 hours under the sameconditions.

FIG. 2( a) shows the C-terminal sequences of melittin and melittin-likepeptides; FIG. 2( b) illustrates that the ε-amino groups of the twolysines are coupled with two glutamate residues to neutralize thepositive charges of the terminus.

FIG. 3 demonstrates using an ATP depletion assay that human prostatecancer cells (LNCaP) are completely lysed after 1 hour in 50 or 100 μMamoebapore helix 3-γ-Glu-γ-Glu, while mouse L-cells remain unaffected.

FIG. 4 shows dose responses of various tumors in vitro to an inventivemasked melittin analog. The numbers 1, 2, 3 and 4 represent 1, 10, 50and 100 μM analog, respectively.

FIG. 5 shows that an inventive melittin analog selectively lysesPSMA-expressing and cancer cells, but does not lyse cells that do notexpress PSMA or a similar molecule.

FIG. 6 indicates that prostate tumors treated withmelittin-gamma-glutamate are growth-inhibited compared to the untreatedcontrol.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compositions and methods for selectivelydestroying a target cell, like a cancer cell. The composition comprisesa procytotoxin that can be activated in a target cell-specific manner,thereby killing, destroying or eliminating the target cell. Methods formaking such procytotoxins are also described.

A procytotoxin according to the present invention is a compound that isnot itself cytotoxic but may be converted into a cytotoxin. A cytotoxinis a substance that is harmful, destructive or deadly to a cell. Apreferred cytotoxin according to the present invention kills orotherwise eliminates the target cell from a patient with high potency.

The pharmaceutical composition according to the instant inventiongenerally does not affect non-target cells. In a preferred embodiment,target cell-specific activation of the procytotoxin occurs only at ornear the target cells. When the pharmaceutical composition is used fortreating cancer cells, the procytotoxin remains in an inactive state,thus is nontoxic, until it reaches the cancer site. In the case ofcancer treatment, the procytotoxin is activated at the surface of thecancer cells and, thereby, it achieves high specificity in thedestruction of target cells versus normal cells. Once the procytotoxinis activated in a target cell-specific manner, the high potency of thecytotoxin ensures that the pharmaceutical composition achieves athorough destruction of the target cells.

Cytotoxic Peptides

According to one embodiment of the invention, a procytotoxin is acytotoxic peptide that is rendered non-toxic by modification of itsmolecular structure, as detailed below. Many naturally occurring andsynthetic cytotoxic peptides are known in the art. Some are useful astherapeutic agents against pathogenic bacteria and other classes ofmicroorganisms; they may be isolated from insects, frogs and otheranimals. Specific examples include alamethicins, attacins, bactenecins,cecropins (see Table 1 the amino acid sequences of cecropins A, B andC), CytA and CytB of Bacillus thurigiensis, defensins, enterocin L50(pediocin L50), lantibiotics, magainins, PGLa, protegrins, sapecin, andsarcotoxin.

In another embodiment of the present invention, the procytotoxin is acytotoxic peptide that is rendered non-toxic by sterically inhibitingformation of an active conformation which renders the peptide toxic.Specifically, the procytotoxin comprises a cytotoxic peptide bound to aninactivator via a peptide bond, wherein said peptide bond is susceptibleto cleavage by a targeting specific protease. The inactivator of theinstant invention hinders the cytotoxic peptide from forming an activeconformation. For example, when the procytotoxin contacts a target cell,a targeting specific protease cleaves the inactivator, thereby allowingthe cytotoxic peptide to aggregate with other cytotoxic peptides intheir active conformation and form a pore to disrupt a cell membrane.

A preferred cytotoxic peptide is a cytolytic peptide. Cytolyticpeptides, also known as channel-forming peptides, typically disrupt cellmembranes, causing cell lysis and death upon contact. Many naturallyoccurring cytolytic peptides from microorganisms, from insects and fromhigher animals are generally known. They often are called hemolysinsbecause they lyse red blood cells as well as other eukaryotic cells.These toxins include Ae I and other cytolysins of sea anemone,aerolysin, amatoxins, amoebapores, amoebapore homologs from Entamoebadispar, brevinin-1E, brevinin-2E, barbatolysin, cytolysin ofEnterococcus faecalis, delta hemolysin, diphtheria toxin, E1 Torcytolysin of Vibrio cholerae, equinatoxins, enterotoxin of Aeromonashydrophila, esculentin, granulysin, haemolysin of Vibrioparahaemolyticus, intermedilysin of Streptococcus intermedius, thelentivirus lytic peptide, leukotoxin of Actinobacillusactinomycetemcomitans, magainin, melittin, membrane-associatedlymphotoxin, Metenkephalin, neokyotorphin and neokyotorphin fragments(1-4), NK-lysin, paradaxins, perforin (especially its amino terminus),perfringolysin O (PFO or theta-toxin) of Clostridium perfringens,phallolysins, phallotoxins and streptolysins. Some hemolysins likemelittin are also known to kill bacteria.

Other cytolytic peptides include D,L-α-amino acid cyclic peptidesselected for lytic activity against mammalian cells. Fernandez-Lopez etal., Nature, 412:452-455 (2001). For instance, attaching a D,L-α-aminoacid cyclic peptide to a microbead will confer a non-toxic procytotoxinconformation. Accordingly, the procytotoxin may have the followingstructure: D,L,-α-amino acid peptide-MMP cleavagesite-Lys-[ε-γ]-Glu-microbead, wherein the [ε-γ] represents a peptidebond between the epsilon amino group of lysine and the gamma carboxylgroup of the adjacent glutamate. In this example, a target cell thatcontains a glutamate carboxypeptidase can cleave gamma glutamate peptidebonds.

Preferably, the cytolytic peptide is a pore-forming cytolytic peptide.Many cytolytic peptides are pore-forming toxins, belonging to a group ofcytotoxins that associate with cell membranes, either nonspecifically orto specific receptors, and form transmembrane pores of discrete size.Most toxic pore-forming peptides employ common features for their celllysis activity. For example, a great number of these toxins lyse cellsthrough a “barrel-stave” mechanism, in which monomers of the toxin bindto and insert into the target membrane and then aggregate like barrelstaves surrounding a central, water filled pore. This pore causes rapidand irreversible electrolyte imbalance of the target cell leading to itsdestruction.

Most pore-forming peptides that act on both mammalian and bacterialcells require an amphipathic alpha-helical structure and a net positivecharge for their cytolytic activity. A strong electrostatic interactionbetween the cationic portion of the peptide and the lipid headgroupsweakens the membrane, facilitating insertion of the hydrophobicalpha-helical peptides. Accordingly, a particularly preferred cytotoxicpeptide according to the invention is a cytolytic, linear α-helicalpeptide. Generally these cytotoxic peptides, in their native form, willhave a net positive charge, which contributes to their cytolyticactivity.

According to a preferred embodiment of the invention, the cytolyticpeptide is melittin or an analog or derivative thereof. Melittin isisolated from bee venom and is a 26 amino acid amphiphilic alpha-helix(Blondelle et al., (1991) Biochemistry 30: 4671-4678; Dempsey et al.,(1991) FEBS Lett. 281: 240-244.) The amino acid sequence of melittin isshown in Table 1. Residues 1-20 are predominantly hydrophobic andresidues 21 to 26 are hydrophilic and basic. Melittin has antibioticactivity, but in mammals it is lytic for leukocytes, red blood cells anda wide variety of other cells. Compounds similar to melittin, which arealso within the scope of the invention, include bombolitin frombumblebee venom (17 amino acid amphiphilic alpha-helix), mastoparan fromwasp venom (14 amino acid amphiphilic alpha-helix) and crabrolin fromhornet venom (13 amino acid amphiphilic alpha-helix) Argiolas A. andPisano J. J., 1985, J. Biol. Chem. 260, 1437-1444.).

TABLE 1 Amino Acid Sequence of Selected Cytolytic Peptides AmoebaporeHelix 3 (Entamoeba histolytica)NH₂-Gly-Phe-Ile-Ala-Thr-Leu-Cys-Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp-Lys-Leu-Ile-Gln-Leu-Ile-Glu-Asp-Lys-CONH₂ (SEQ ID NO: 5) Cecropin A(Antheria pernyi)NH₂-Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Glu-Lys-Val-Gly-Gln-Asn-Ile-Arg-Asp-Gly-Ile-Ile-Lys-Ala-Gly- Pro-Ala-Val-Ala-Val-Val-Gly-Gln-Ala-Thr-Gln-Ile-Ala-Lys-COOH (SEQ ID NO: 6) Cecropin B (Antheria pernyi)NH₂-Lys-Trp-Lys-Ile-Phe-Lys-Lys-Ile-Glu-Lys-Val-Gly-Arg-Asn-Ile-Arg-Asn-Gly-Ile-Ile-Lys-Ala-Gly-Pro-Ala-Val-Ala-Val-Leu-Gly-Glu-Ala-Lys-Ala-Leu-COOH (SEQ ID NO: 7) Cecropin D (Antheria pernyi)NH₂-Trp-Asn-Pro-Phe-Lys-Glu-Leu-Glu-Lys-Val-Gly-Gln-Arg-Val-Arg-Asp-Ala-Val-Ile-Ser-Ala-Gly-Pro-Ala-Val-Ala-Thr-Val-Ala-Gln-Ala-Thr-Ala-Leu-Ala-Lys-COOH (SEQ ID NO: 8) Melittin (Apis mellifera)NH₂-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-COOH (SEQ ID NO: 9)

A particularly preferred pore-forming peptide, according to the instantinvention, is amoebapore, a 77-residue pore-forming peptide from theamoebae Entamoeba histolytica (Young et al., (1982) J. Exp. Med. 156:1677; Lynch et al., (1982) EMBO J 7: 801; Young & Cohn, (1985) J. CellBiol. 29: 299; Rosenberg et al., (1989) Molec. Biochem. Parasit. 33:237; Jansson et al., (1994) Science 263: 1440). It has four alphahelices, from amino acids approximately 1-21, 25-36, 40-63 and 67-77,conventionally called helices 1, 2, 3, and 4, respectively.

Three isoforms of amoebapore are known: amoebapore A, B and C,respectively. This peptide is stabilized by three disulfide bonds andcontains four mostly amphipathic alpha-helical structures. The thirdamphipathic helical structure (helix 3) retains the cytolytic activitysimilar to the wild type peptide. A synthetic peptide based on thesequence of its third amphipathic alpha helix have recently been shownto have cytolytic activity for nucleated cells at high concentrations(10-100 μM) (Leippe et al., (1994) Proc. Natl. Acad. Sci. USA 91: 2602).Accordingly, a particularly preferred cytotoxin is a derivative of theamoebapore cytolytic peptide listed in Table 1:NH₂-Gly-Phe-Ile-Ala-Thr-Leu-Cys-Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp-Lys-Leu-Ile-Gln-Leu-Ile-Glu-Asp-Lys-CONH₂(SEQ ID NO: 5).

It is readily recognized that the above described cytolytic peptides maybe modified or derivatized to produce analogs and derivatives whichretain, or even exhibit enhanced, cytolytic activities. For example,Andra et al. disclose that shortened amoebapore analogs have enhancedantibacterial and cytolytic activities (FEBS Letters, 385 (1996), pp.96-100, incorporated herein by reference in its entirety).

In designing such analogs or derivatives, the artisan will be informedby the foregoing discussion relating to the amphipathic alpha-helicalstructure and net positive charge that are implicated in the cytolyticactivity. Thus, a skilled artisan is able to design amoebapore analogs,i.e., non-native forms never before known in nature, based on theobserved homologies and known structure and properties of the nativeprotein, to be used as a cytolytic peptide in accordance with theinstant invention.

Modification and derivatization according to the instant inventioninclude, but are not limited to, substitutions, additions or deletionsthat provide for functionally equivalent molecules. (Function may beassessed in accord with the working examples presented below.) Analogsand derivatives may also be made via modifications of side chains ofamino acid residues of the cytotoxic peptides, preferably, with enhancedor increased functional activity relative to the native protein orpolypeptide.

For example, analogs and derivatives of an amoebapore or other cytolyticpeptides include, but are not limited to, those containing, as a primaryamino acid sequence, all or part of the amino acid sequence the nativepeptide, such as with altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a conservative amino acid substitution. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity, which acts as a functionalequivalent, resulting in a silent alteration. Substitutes for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. Polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. Positively charged (basic) amino acids include arginine,lysine and histidine. Negatively charged (acidic) amino acids includeaspartic acid and glutamic acid. Because the terminal positive chargesare thought to be involved in the cytolytic activity of the peptide,addition or substitution of a positively charged amino acid at theC-terminus is particularly contemplated to enhance its cytolyticpotency.

Dimerization, truncation, diasteroisomers (D-amino acid-incorporatedanalogs) (Shai et al., (1996) J. Biol. Chem. 271-7305-7308), andcombinations thereof may also be employed for producing derivatives andanalogs of pore-forming peptides. For example, Werkmeister et al.(1993), Biochim. Biophys. Acta 1157: 50-54, discloses the effect ofsequence and structural variations on the cytolytic activity ofmelittin, and is hereby incorporated by reference in its entirety.

The cytolytic peptides of the invention will normally contain from about15 to about 40 amino acid residues. It is apparent to one skilled in theart that active low molecular weight peptides containing less than about40 amino acids (or even less than about 30) are normally not difficultto synthesize chemically, while peptides with more than about 40 aminoacids are relatively difficult to synthesize in pure form by chemicalmethods, and may be best prepared by fermentation or by recombinant DNAprocedures from the appropriate genetic coding sequence.

A particular advantage of the useful peptides of this invention is thatthey are readily synthesized by solid phase methods and a variety ofcombinations are possible to achieve specifically required results. Anadvantage of the use of solid phase techniques is that the product canbe directly synthesized with the C-terminus amidated or otherwiseblocked, which is beneficial in forming the procytotoxins of theinvention. For example, one procytotoxin requires formation ofγ-glutamate peptide bonds which are readily produced by such chemicalsynthesis.

Inactivator

An inactivator according to the present invention is an object that,when attached to a lytic peptide, inactivates the cytotoxin by chargeneutralization and/or sterically inhibiting formation of a conformationwhich renders the peptide toxic. The inactivator, for example, may be aphage via phage engineering or a streptavidin microbead via peptidebiotinylation. Therefore, the procytotoxin may comprise the followingstructure: lytic peptide-MMP2 cleavagesite-Lys-[ε-γ]-biotin-streptavidin coated microbead. Accordingly, cellswith a matrix metalloprotease (specifically, MMP-2) will cleave theprocytotoxin at the MMP-2 cleavage site. The lytic peptide can now foldinto its active conformation and aggregate with other cytolytic peptidesto form a pore and lyse the target cell. The inactivator may also be anegatively charged amino acid, such as glutamic acid or aspartic acid.

The inactivator may also be an amino acid, but is typically a peptide.The peptide may vary in size and in fact, need not be “big”. So long asthe amino acids charge neutralize and/or create steric restriction oflytic peptide membrane insertion and organization into a pore, thepeptide is a suitable inactivator.

The inactivator can be added to the N- or C-terminus of the cytotoxicpeptide, or both. In a preferred embodiment, the cytotoxic peptide is acytolytic peptide, and the inactivator is added to the C-terminus of acytolytic peptide. Furthermore, the inactivator may additionallycomprise a targeting molecule, as discussed below.

Targeting Specific Protease

A targeting specific protease as described herein is an enzymeassociated with a target cell that cleaves a peptide bond of proteins. Atarget cell, as described herein, may be any cell whose lysis isdesired. For example, a target cell could be a cancer cell, a bacterialcell, or endothelial cells of the tumor neo-vasculature.

The targeting specific protease of the present invention may betypically expressed in tumors, but need not be exclusively expressed intumors. Accordingly, the targeting specific protease may bepreferentially expressed in tumor cells when compared to normal cells.Additionally, the targeting specific protease may be a proteaseexpressed by a non-tumorigenic target cell, such as a bacterial cell,but is nevertheless preferentially expressed by a target cell whencompared to a non-target cell. Accordingly, the procytotoxin asdescribed herein is additionally useful as an antimicrobial orantibacterial agent.

In a preferred embodiment, the targeting specific protease is MMP. Stillpreferred, the targeting specific protease is MMP-2. MMPs are associatedwith angiogenesis, as well as neo-vessel formation. Therefore, targetingthe vasculature which supplies the tumors, as well as newly formedvessels, prevents tumors from forming.

For example, lytic peptides of the instant invention linked to aninactivator by a sequence of amino acids recognized by MMP can beselectively released from the inactivator in the presence of a targetingspecific protease such as MMP. The cytotoxic peptide is then free toform its active conformation and lyse target cells.

Additional reactive peptide substrates suitable for the presentinvention include those described for PSA in Coombs et al., Chem Biol,5(9):475-88 (1988), PSMA and gamma-glutamyl hydrolase (GGH).

Targeting Molecule

The targeting molecule of the instant invention is an optional featurethat presents an extra measure of selectivity. The targeting moleculedirects the procytotoxin to the target cell, where the procytotoxin isrendered toxic and selectively lysis its target. Additionally, thetargeting molecule may act as an inactivator of the cytolytic peptide.In this regard, the targeting molecule may charge neutralize orsterically inhibit a cytolytic peptide from pore formation. Thetargeting molecule may be added to the N- or C-terminus, or both.

In a preferred embodiment, the targeting molecule is an antibody.Preferably, the antibody is an anti-fibronectin ED-B antibody, therebydirecting the procytotoxin to the extracellular matrix associated withneo-vessel formation. The antibody in the instant invention can be afull-length (i.e., naturally occurring or formed by normalimmunoglobulin gene fragment recombinatorial processes) immunoglobulinmolecule (e.g., an IgG antibody) or an immunologically active (i.e.,specifically binding) portion of an immunoglobulin molecule.

Antibody fragments which recognize specific epitopes can be generated byknown techniques. For example, such fragments include, but are notlimited to: the F(ab)′₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab′ fragments, which can begenerated be generated by reducing disulfide bridges of the F(ab)′₂fragments. Alternatively, Fab′ expression libraries can be constructed(Huse et al., 1989, Science, 246:1274-1281) to allow rapid and easyidentification of monoclonal Fab′ fragments with the desiredspecificity.

The antibodies described herein can be polyclonal or monoclonalantibodies. Polyclonal antibodies are heterogeneous populations ofantibody molecules derived from the sera of animals immunized withantigen, such as a target gene product, or an antigenic functionalderivative thereof. Monoclonal antibodies are homogeneous populations ofantibodies to a particular antigen and the antibody comprises only onetype of antigen binding site to which the nucleic acid specificallybinds.

Targeting molecules that target the neo-vasculature can also be added tothe inactivator. Molecules that target the neo-vasculature can easily beidentified by screening phage display libraries. Any such peptide wouldbe a suitable targeting molecule of the present invention.

Targeting molecules which bind specifically to integrins are one classof molecules that target the neovasculature. These peptides bear thesequence based on Arg-Gly-Asp (RGD). Accordingly, sequences that bindcertain integrins can serve as a useful targeting molecules toendothelial cells and other cells of the neo-vasculature. In a preferredembodiment, the targeting molecule is an RGD targeting sequence.

Non-structural spacers may be a feature of the targeting molecule. Suchspacers typically comprise glycine and/or proline residues. Preferablylengths of these spacers range from about one to about 5 amino acids,with two being particularly preferred. In addition, it is oftenpreferable to physically constrain the targeting molecule bycyclization, which usually results in increased binding. This is usuallyaccomplished by a pair of cysteine residues, flanking the RGD core at adistance of about 4 (having only RGD in between) to 10 amino acids fromone another, and preferably about 7 amino acids from one another.

Thus, a typical targeting molecule would have the following structure:-XRGDYX-wherein X is zero to five amino acids and Y is a one or two amino acids,selected from cysteine, serine, threonine and methionine. In aparticularly useful embodiment, X is comprised of glycine residues, butoptionally contains at least one, and typically one or two, free thiol-or amine-containing amino acids and/or a single hydrophobic amino acid.Thiol-containing residues include methionine and cysteine;amine-containing residues include lysine and (at least one additional)arginine; and hydrophobic residues include leucine, isoleucine, alanineand phenylalanine.

Unless otherwise indicated by context, the term “RGD” refers not only tothe peptide sequence Arg-Gly-Asp, it refers generically to the class ofminimal or core peptide sequences that mediate specific interaction withintegrins. Thus, an “RDG targeting sequence” encompasses the entiregenus of integrin-binding domains.

Procytotoxins

In order to enhance its therapeutic usefulness, a cytotoxin is modifiedaccording to the invention to its non-toxic form, known as aprocytotoxin. Accordingly, a method for making a procytotoxin,comprising modifying a cytotoxic peptide to include an inactivator, isdescribed.

Numerous methods of modifications are available, the applicability ofwhich will depend on the structural characteristics of the cytotoxin. Apeptide, for example, may be added to either the N-terminus and/orC-terminus of the cytotoxic peptide, such that the cytotoxic peptide issterically hindered and therefore rendered non-toxic. Stericallypreventing the alpha-helical structure of the cytotoxin from forming orinhibiting the organization of multiple proteins required for poreformation, is a preferred embodiment. Additionally, the cytotoxicpeptide can be modified to include negatively charged amino acids,thereby preventing aggregation of multiple proteins into a toxic poreconformation.

The procytotoxin of the present invention comprises a cytotoxic peptidebound to an inactivator via a peptide bond, wherein said peptide bond issusceptible to cleavage by a targeting specific protease. Preferably,the cytotoxic peptide is a pore-forming cytolytic peptide, as describedabove. Still preferred, the cytolytic peptide is a melittin, a melittinanalog, or a melittin derivative.

For instance, the procytotoxin of the present invention comprises thefollowing structure:Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Gly-Ala-Ile-Gly-Gln-Pro]-(X)(SEQ ID NOS 1 & 2, respectively), wherein (X) is an inactivator and thepeptide marked in brackets can be oriented in either direction. Theinactivator can be selected from the group consisting of a microbead, anamino acid, a peptide, phage and a phage filament. Cleavage by MMP atthis peptide will yield a melittin peptide with few additional aminoacids on the C-terminus (Gly-Ala-lle) which should not interfere withpore formation.

In a related vein, the procytotoxin of the present invention comprisesGly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Ser-Ser-Phe(or Tyr)-Tyr-Ser-Gly(or Ser)]-(X) (SEQ ID NOS 3 & 4,respectively), wherein (X) is an inactivator and the peptide marked inbrackets can be oriented in either direction. Cleavage of this peptidewith PSA will render the peptide toxic.

Also contemplated in the instant invention is a targeting molecule thatadds an additional-measure of selectivity. For example, the procytotoxinmay comprise the following structure: lyticpeptide-[Gln-Gly-Ala-Ile-Gly-Gly-Pro]-Lys-[ε-γ]-biotin- (SEQ ID NO: 10)streptavidin coated microbead-RGD targeting sequence.

In addition to directing the procytotoxin in or around the cell, thetargeting molecule may act as an inactivator as well, maintaining thecytolytic peptide in its inactive state. A targeting molecule of theinstant invention may be selected from the group consisting of amolecule that targets the neovasculature and an antibody. In a preferredembodiment, the targeting molecule is an RGD targeting sequence.

Furthermore, the procytotoxin may also comprise at least one lysineresidue bound via a peptide bond to at least one amino acid via theε-amino group of said lysine residue. The inventors have recognized thata common feature of some of the aforementioned cytotoxins is thepresence of one or more lysine residues. Accordingly, a preferred meansof preparing a protoxin is to modify one or more of these lysineresidues. Thus, for example, by addition via a peptide bond of anegatively charged inactivator, such as an amino acid, to the epsilonamino group of lysine, charge neutralization and/or steric mechanismsare invoked to maintain the toxin as a protoxin. Of course, where anative molecule lacks a lysine, it may be added and modified, whichstill is expected to invoke the steric inhibition of toxin formation.Generally, the amino acid added to the epsilon amino group using apeptide linkage will be the target of a membrane-associated protease. Anincreasing number of such enzymes are being identified which arecorrelated with neoplastic or preneoplastic states. Thus, they areconvenient targets for therapy.

In one aspect protoxin formation is accomplished via the conversion ofan epsilon amino group of a lysine residue to a neutral peptide linkageand/or the addition of one or more negatively charged amino acidresidues at or near this position. A preferred negatively charged aminoacid is glutamate. In a particularly preferred embodiment the epsilonamino group of lysine is bound by a peptide linkage to the gammacarboxyl group of glutamate. The resultant free alpha carboxyl group insuch a case is free to neutralize additional adjacent positively chargedamino acids, like arginine, which is expected to further repress theactivity of the cytotoxin. In some embodiments it is envisioned thatmore than one glutamic acid (poly-α-γ-glutamate) is added to a lysine.Thus, a peptide lysine is bound via an ε-γ linkage to a glutamate andthat glutamate is bound to a second glutamate via an α-γ peptide bond.The second glutamate may be bound to a third, and so on.

It will be apparent to the artisan, therefore, that a more generalaspect of the invention entails adding any amino acid to partially orwholly neutralize the subject positive charge, or otherwise tosterically inhibit formation of the toxin. For example, where a lysineresidue is present, as described above, any amino acid may be linked tothe epsilon amino group, via a linkage with the alpha carboxyl group ofthe added amino acid. In general, a salient feature is the ability ofthe added amino acid to be proteolytically cleaved in order to formtoxin from protoxin. Thus, in the case of chymotrypsin-like activity,the bulky hydrophobic amino acids, like tyrosine, phenylalanine andtryptophan may be employed. In a case where steric considerations aloneare sufficient to inactivate the toxin, positively charged amino acidslike arginine and lysine may be employed to invoke trypsin-likeactivity. Other examples also are apparent based on the artisan'sknowledge of protease specificity's.

Particularly preferred procytotoxins include amoebapore, its analogs andits derivatives that contains one or more y-linked glutamate residueslinked via a peptide bond to the epsilon amino group of at least onelysine, preferably the C-terminal-most lysine (hereinafter“γ-glutamate-masked amoebapore analog”). A particularly preferredprocytotoxin has the following structure:Gly-Phe-Ile-Ala-Thr-Leu-Cys-Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp-Lys-Leu-Ile-Gln-Leu-lle-Glu-Asp-Lys-[α-γ]-Glu(SEQ ID NO: 11), wherein [α-γ] represents a peptide bond between theepsilon amino group of lysine and the gamma carboxyl group of theadjacent glutamate and [α-γ]represents a peptide bond between the alphaamino group of the first glutamate and the gamma carboxyl group of thesecond glutamate.

In addition, amoebapore and other cytotoxic peptides can be modifiedwith other amino acids. One such exemplary protoxin has the followingstructure:Gly-Phe-Ile-Ala-Thr-Leu-Cys-Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp-Lys-Leu-Ile-Gln-Leu-Ile-Glu-Asp-Lys-[ε-α]-Phe(SEQ ID NO: 12), wherein [ε-α]represents a peptide bond between theepsilon amino group of lysine and the alpha carboxyl group of theadjacent phenylalanine. Another exemplary protoxin that can be activatedby chymotrypsin-like activity has the following structure:Gly-Phe-Ile-Ala-Thr-Leu-Cys-Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp-Lys([ε-α]-Phe)-Leu-Ile-Gln-Leu-Ile-Glu-Asp-Lys-[ε-α]-Phe(SEQ ID NO: 13), using the same nomenclature and whereLys([ε-α]-Phe)-Leu represents a linkage between the epsilon amino groupof lysine and the alpha carboxy group of phenylalanine, and a standardpeptide linkage between lysine and phenlyalanine. Of course, thephenylalanine may be replaced with other amino acids, such as tyrosineand tryptophan in the case of chymotrypsin-like activity. In someinstances, in order to invoke trypsin-like activity, it may bebeneficial to utilize positively charged amino acids, like arginine andlysine, instead of phenylalanine.

Other particularly preferred procytotoxins include melittin, its analogsand its derivatives that contain at least one γ-linked glutamate residuelinked via a peptide bond to the epsilon amino group of a lysine(hereinafter “γ-glutamate-masked melittin analog”). As indicated inTable 1, melittin has two lysines and two adjacent arginines near itsC-terminus. When one of the lysines is so masked, it is expected thatthe free alpha carboxyl group would act to neutralize the adjacentarginine, further contributing to the inhibition of the toxic activityof melittin. A particularly preferred procytotoxin has the followingstructure:Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys([ε-γ]-Glu)-Arg-Lys([ε-γ]-Glu)-Arg-Gln-Gln(SEQ ID NO: 14), wherein -Lys-([ε-γ]-Glu)-Arg- represents a peptide bondbetween the epsilon amino group of lysine and the gamma carboxyl groupof the adjacent glutamate and a standard peptide bond between the lysineand arginine residues. Of course, -Lys-([ε-γ]-Glu)-Arg- can be replaced,for example, by -Lys([ε-α]-Phe)-Leu-, as detailed above, andphenylalanine can be replaced by other amino acids like tyrosine andtryptophan to invoke chymotrypsin-like activity. In some instances, whentrypsin-like activity is being invoked, it may be beneficial to utilizepositively charged amino acids, like arginine and lysine, instead ofphenylalanine in this latter example.

With regard to the terminology used herein, the artisan will recognizethat a “standard” peptide bond is formed between the alpha carboxylgroup of one amino acid with the alpha amino group of the next aminoacid in the peptide chain and that peptide sequences are read from theiramino-terminal end to their carboxyl-terminal end. For clarification,the following structures of glutamic acid and lysine are presented withthe various groups named according to which carbon of the amino acidbackbone they attach:

A set of particularly preferred procytotoxins have the followingstructures: (1)Gly-Phe-Ile-Ala-Thr-Leu-Cys-Thr-Lys(R)-Val-Leu-Asp-Phe-Gly-Ile-Asp-Lys(R)-Leu-Ile-Gln-Leu-Ile-Glu-Asp-Lys(R)(SEQ ID NO: 15), and (2) Gly-Ile-Gly-Ala-Val-Leu-Lys(R)-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys(R)-Arg-Lys(R)-Arg-Gln-Gln(SEQ ID NO: 16), wherein R is independently selected from the groupconsisting of the ε-amino group of the adjacent lysine residue,[ε-γ]-Glu, [ε-γ]-Glu-[α-γ]-(Glu)₁₋₃, [ε-α]-(Phe)₁₋₃, [ε-α]-(Tyr)₁₋₃,[ε-α]-(Trp)₁₋₃, [ε-α]-(Lys)₁₋₃ and [ε-α]-(Arg)₁₋₃, wherein [ε-γ]represents a peptide bond between the epsilon amino group of lysine andthe gamma carboxyl group of the adjacent glutamate, [α-γ] represents apeptide bond between the alpha amino group of the first glutamate andthe gamma carboxyl group of the second glutamate, [ε-α] represents apeptide bond between the epsilon amino acid of lysine and the alphacarboxyl group of the indicated amino acid and the subscript indicatesthat additional numbers of the designated amino acid can be linked tothe first via conventional peptide bonds. With regard to the subscriptednumbers, it is understood that larger numbers of amino acids arepossible, e.g., 4, 5, 6, etc., but 1, 2, and 3 are anticipated to beoptimal.

It is therefore also understood that in addition to or instead of stericdeterminants, charge determinants are also important. Where the positivecharge of the pore forming peptide is involved in its cytolyticactivity, eliminating/neutralizing the charge renders the peptidenon-toxic. It is envisioned that even a partial neutralization of thispositive charge will be effective. Accordingly, disrupting the alphahelical structure of the cytotoxin by sterically preventing thestructure from forming in addition to eliminating/neutralizing thepositive charge of the pore-forming peptide is also preferred. Thus,even if no neutralization is accomplished, some of the modificationsbelow will result in steric alterations which will inactivate the toxinto create the protoxin.

While not wishing to be bound by any theory, the inventors recognize theimportance of the positive charges for the mode of action of theamoebapore and related cytotoxic molecules, and believe that theglutamate residues added according to the invention neutralize andreverse the polarity of these positive charges of a cytolytic peptide orits analog or derivative, thus negating its cell-lysing activities. Inthe case of other amino acids, they may function by a partialneutralization. Of course, steric determinants also are believed to beimportant in maintaining the toxins in protoxin form in many, if not allcases.

The procytotoxic peptides of this invention may be chemicallysynthesized by standard solid phase procedures using the protection,deprotection and cleavage techniques and reagents appropriate to eachspecific amino acid or peptide. A combination of manual and automated(e.g., APPLIED BIOSYSTEM.® 430A) solid phase techniques can be used tosynthesize the novel peptides of this invention. For background on solidphase techniques, reference is made to Andreu et al., (1983) Proc. Natl.Acad. Sci USA 80: 6475-6479; Andreu et al., (1985) Biochemistry 24:1683-1688; Fink et al. (June 1989) Int. J. Peptide Protein Res. 33:412-421; Fink et al., (1989) J. Biol. Chem. 264: 6260-6267; each ofwhich is incorporated herein by reference.

The in vivo stability of the procytotoxin of the invention can beimproved by adding a D-amino acid to the N- or C-terminus, whicheverdoes not have a γ-linked glutamic acid residue. (Of course, some in vivoinstability would be advantageous because it would decrease the chanceof possible adverse side effects that might arise once the procytotoxinis converted to cytotoxin.) This procedure is particularly useful withproducts of the invention which are employed under conditions,parenteral or oral, where they will be subject to hydrolysis bynaturally occurring enzymes before they perform their desiredphysiological function.

Pharmaceutical Compositions

Another aspect of the present invention is a pharmaceutical compositioncomprising one or more procytotoxins of the invention and apharmaceutically suitable carrier or excipient.

While a procytotoxic peptide of the present invention can beadministered, alone, to a patient, it is preferable to present thepeptide as part of a pharmaceutical formulation. It is also preferred toadminister more than one procytotoxin, or combinations of procytotoxinsas part of a pharmaceutical formulation. Pharmaceutically suitableexcipients typically include carriers known to those skilled in the art,including pharmaceutical adjuvants. Generally, these pharmaceuticallyacceptable carriers will include water, saline, buffers, and othercompounds described, e.g., in the MERCK INDEX, Merck & Co., Rahway, N.J.See also Bioreversible Carriers in Drug Design, Theory and Application,Roche (ed.), Pergamon Press, (1987). These formulations typicallycomprise the pharmacological agent (i.e., the procytotoxin) in atherapeutically or pharmaceutically effective dose together with one ormore pharmaceutically or therapeutically acceptable carriers andoptionally other therapeutic ingredients, Various considerations aredescribed, e.g., in Gilman et al. (eds) (1990) Goodman and Gilman's: ThePharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; NovelDrug Delivery Systems, 2nd Ed., Norris (ed.) Marcel Dekker Inc. (1989),and Remington's Pharmaceutical Sciences, the full disclosures of whichare incorporated herein by reference.

The compositions may be formulated in any pharmaceutical formappropriate for the desired route of administration. Examples of suchcompositions include solid compositions for oral administration such astablets, capsules, pills, powders and granules which may be entericcoated or otherwise protected from hydrolysis, especially enzymatichydrolysis, liquid compositions for oral administration such assolutions, suspensions, syrups or elixirs and preparations forparenteral administration such as sterile solutions, suspensions oremulsions. The compositions may also be manufactured in the form ofsterile solid compositions which can be dissolved in sterile water,physiological saline or some other sterile injectable medium immediatelybefore use.

Since the procytotoxins of the invention are amphoteric they may beutilized as free bases, as acid addition salts or as metal salts. Thesalts must, of course, be pharmaceutically acceptable, and these willinclude metal salts particularly alkali and alkaline earth metal salts,suitably potassium or sodium salts. A wide variety of pharmaceuticallyacceptable acid addition salts are available. These include thoseprepared from both organic and inorganic acids, preferably mineralacids. Typical acids which may be mentioned by way of example includecitric, succinic, lactic, hydrochloric and hydrobromic acids. Suchproducts are readily prepared by procedures well known to one skilled inthe art.

In all such compositions, the cytolytic peptides will normally be theprincipal physiologically active ingredient. The inventive peptides maybe formulated, however, with additional pharmacological agents forcombination therapies. When used in treating cancer, for example, theymay be formulated with compatible conventional chemotherapeutic agents.

Methods for administration are also discussed in Gilman et al. (eds)(1990) and Norris (ed.), supra. In particular, the pharmaceuticalcomposition of the invention may be administered intravenously,subcutaneously, orally, transdermally, such as in the method of(Prausnitz, M R, Bose, V G, Langer, R, Weaver, J C; Electroporation ofMammalian skin: a mechanism to enhance transdermal drug delivery. Proc.Nat'l. Acad. Sci. U.S.A., 90:10504-10508 (1993); and Wallace, B M,Lasker, J S: Stand and Deliver: getting peptide drugs into the body.Science 260:912-912 (1992). Liposomes may also be used to administer theprocytotoxin of the invention (see Woodle, M C, Storm, G, Newman, M S,Jekot, J J, Collins, L R, Martin, F J, Szoka F C: Prolonged systemicdelivery of peptide drugs by long circulating liposomes: illustrationwith vasopressin in the Brattleboro rat. Pharmaceut. Res. 9:260-265(1992).

Therefore, a method for treating a cancer patient, comprisingadministering a therapeutically effective amount of the pharmaceuticalcompositions of the instant invention is described. More than oneprocytotoxin may be administered, and combinations of procytotoxins mayalso be administered. Optimal delivery routes, dosages and regimens fora given mammalian host can be readily ascertained by one skilled in theart. It will, of course, be appreciated that the actual dose used willvary according to the particular composition formulated, the particularcompound used, the mode of application and the particular site, host anddisease being treated. Many factors that modify the action of the drugwill be taken into account including age, weight, sex, diet, time ofadministration, route of administration, rate of excretion, condition ofthe patient, drug combinations, reaction sensitivities and severity ofthe disease.

Method of Selectively Destroying a Target Cell

The peptide protoxin of the present invention typically is converted,and thereby activated, into a cytotoxic peptide in a targetcell-specific manner by an activity associated with the target cell. Inother words, the target cell possesses a specific mechanism forconverting the procytotoxin into a cytotoxin. The activated cytotoxicpeptide assembles with other cytotoxic peptides and destroy a targetcell in a selective manner. Most conveniently, this cell-associatedactivity is a protease.

Accordingly, a method for selectively destroying a target cell,comprising contacting the target cell with procytotoxin, which comprisesa cytotoxic peptide bound via a peptide bond to an inactivator, whereinsaid peptide bond is susceptible to cleavage by a targeting specificprotease, is described. Preferably, the target cell is a cancer cell,but any cell that comprises the targeting specific protease may be atarget.

Preferably, the target cell has a protease that selectively cleaves thepeptide bond to which the inactivator is attached. Therefore, if theprocytotoxin comprises an MMP cleavage site, a target cell with an MMPcan cleave the peptide bond and “activate” the cytolytic peptide. Alsopreferred, the procytotoxin comprises a targeting molecule selected fromthe group consisting of an RGD targeting sequence and a neovasculartargeting sequence of an anti-fibronectin ED-B antibody. These targetingsequences may act as inactivators themselves, as well as direct aprocytotoxin to the neo-vasculature and cytoskeletal elementssurrounding the target cell.

In another preferred embodiment, the procytotoxin of the instantinvention further comprises a cytotoxic peptide having at least onelysine residue bound via a peptide bond to at least one amino acid viathe ε-amino group of said lysine residue. Preferably, the epsilon aminogroup of lysine is bound by a peptide linkage to the gamma carboxylgroup of a glutamate. A target cell that contains a glutamatecarboxypeptidase can cleave gamma glutamate peptide bonds. Consequently,when a γ-glutamate-masked cytolytic peptide is contacted with a targetcell that contains a glutamate carboxypeptidase, the γ-glutamateresidues are removed from the protoxin, and the resultant cytolyticpeptide molecule causes lysis of the target cell. A non-target cell,which does not contain the glutamate carboxypeptidase, is unable toactivate the protoxin and is thus unaffected by the protoxin.

A skilled person would readily recognize that if the glutamate residueis linked to the ε-amino group of a lysine, a carboxypeptidase,including the prostate specific membrane antigen discussed below, wouldbe able to cleave the γ-linked glutamate residues regardless whetherthey are located at the C-terminus or the N-terminus of the cytotoxicpeptide or somewhere in between.

Thus, it will be recognized that such an approach has broaderapplicability to cells associated with a particular activity that can beinvoked to convert the protoxin to its toxin form. For example, when thetarget cell is associated with a chymotrypsin-like activity, theprotoxin can be rendered non-toxic by modification with phenylalanine,tyrosine or tryptophan. Since these enzymes, and indeed serine proteasesgenerally, are known to cleave C-terminally, relative to the amino acidtarget site, when phenylalanine, for example, is linked via an [ε-α]bond to lysine, the protease will cleave after the alpha carbonyl ofphenylalanine, restoring the epsilon amino group of lysine (and thecarboxy group of phenylalanine), thereby activating the toxin. Again,when the target cell has trypsin-like activity, the protoxin may bemodified with a positively charged amino acid like lysine or arginine.

In cases where the target cell has a protease activity that is the sameor similar to conventional proteases, like chymotrypsin and trypsin,there should not be unwanted activation of the protoxin. That is,normally, chymotrypsin and trypsin are localized primarily in theintracellular environment, which is not accessible to the presentprotoxin medicaments. In the case of disorders where the target cellsare associated with elevated levels of outer membrane-associatedproteases, which are contacted with the protoxin due to theirextracellular location, only the diseased cells will have the capabilityof activating the protoxin to its toxic form. Accordingly, the presentprocytotoxins are envisioned as having low levels of toxicity.

The present invention also comprehends a method for the treatment ofcancers. According to an embodiment of the present invention, apharmaceutical composition comprising the inventive protoxin isadministered to a cancer patient, whose cancer cells are target cellspossessing, for example, the requisite glutamate carboxypeptidase (orother enzyme for pro-drug conversion) for activating the procytotoxin.

Therapeutic treatment of cancer using the instant cytolyticpeptide-based procytotoxin is particularly advantageous becausecytolytic peptides are known to be absorbed into the target cellmembrane. Even after the target cell is lysed, therefore, the cytolyticpeptides are prevented from acting on, and causing undesired destructionof, adjacent non-target cells. For example, see Leippe et al. (1991)Proc. Natl. Acad. Sci. 88: 7659-7663). Moreover, at least in the casewhere no extra stabilization steps (described above) are taken, it isnoted that the instant medicaments are small peptides, which generallywill have a fairly short half-life. Hence, even if small amounts ofactivated cytolytic peptide escapes the surface of the cancer cell, itshould be reasonably short-lived and cause little, if any, destructionof non-target cells.

Particularly preferred embodiments of the present invention entail usingγ-glutamate-masked amoebapore or γ-glutamate-masked melittin analogs totreat prostate cancer. It has been recently discovered that prostatecancer cells over-express a type II transmembrane protein, the prostatespecific membrane antigen (“PSMA”). PSMA has an intracellular epitopethat is immunoreactive toward the 7E11C5 immunoglobulin G monoclonalantibody. Horosczewicz et al. (1983), LNCAP model of human prostaticcarcinoma, Cancer Res. 43: 1809-1818. In prostate cancer patients, PSMAis highly expressed on malignant prostate epithelia, but only marginallyon normal prostate glands, and to a lesser degree on benign prostatichypertrophic epithelia. Pinto et al. (1999) Prostate specific membraneantigen, a unique glutamate carboxypeptidase: a review of recentfindings, The Prostate J. 1: 15-26; Wright et al. (1995) Expression ofProstate Specific Membrane Antigen (PSMA) in normal benign and malignantprostate tissues, Urol. Oncol. 1: 18-28; Lopes et al., (1990)Immunohistochemical and pharmacokinetic characterization of thesite-specific immunoconjugate CYT-356 derived from antiprostatemonoclonal antibody 7E11-C5, Cancer Res. 50: 623-6428; Troyer et al.,Detection and characterization of the prostate-specific membrane antigen(PSMA) in tissue extracts and body fluids, Int. J. Cancer 62: 552-558.

The proteolytic domain of PSMA is located on the outside of the cellsurface. Upon reaching the target cell, the procytotoxins of theinvention, without having to be internalized by the target cell, willhave their γ-glutamate residue(s) cleaved and removed, thereby they willbe activated. The procytotoxins thus are activated to cytotoxinsprecisely at the desired site of action, the target cell. The cytotoxinacts directly on the target cell, thereby increasing effectiveness ofprostate cancer cell killing. The activated cytolytic peptideimmediately inserts into the target cell, leaving it almost no chance ofacting upon adjacent, non-target cells. In fact, it is possible that oneend of the peptide is already inserted into the membrane before it isactivated by PSMA. Furthermore, as discussed above, the activated toxinis neutralized by the membrane therefore after the lysis of the targetcell, the cytolytic peptides remain adsorbed in the membrane debris ofthe target cell and do not leak and harm non-target cells.

The inventors also have recognized that a range of cancer cells, beyondthose of prostate specificity, apparently have the ability to cleave thesubject γ-glutamate-masked cytolytic peptides (see Examples). On theother hand, normal cells appear to lack this ability, rendering theinventive compounds highly effective in ablating cancer cells, yetleaving normal cells unaffected. Cancer cell types showing sensitivityto the inventive toxins include prostate, ovary, lung and melanoma. Asearch of public expressed sequence tag databases for PSMA-likemolecules indicated that, whereas a number of cancers cells expressproteins with significant degrees of homology to PSMA, normal cells donot. This observation likely explains the ability of the inventivemolecules to act on a variety of tumor cells, while leaving normal cellsunaffected.

In a further embodiment, the procytotoxin is maintained in inactive formby modification with a bulky hydrophobic amino acid, like phenylalanine,tyrosine and tryptophan, or positively charged amino acids, like lysineand arginine. Such protoxins are useful in treating target cells thatare associated with a chymotrypsin- or trypsin-like activity. Theartisan will be aware of other examples as well. Target cells capable ofactivating such a toxin include pancreatic cancer cells, which areassociated with elevated levels of TMPRSS3, a transmembrane serineprotease. Walltrapp et al., Cancer Res. 60: 2602-06 (2000).

The following non-limiting examples are given by way of illustrationonly and are not to be considered limitations of this invention, andthere are many apparent variations within the scope of this invention.The examples illustrate the in vitro activity of the compounds of thisinvention and are illustrative of the studies that the art has relied onfor close to half a century as reasonably predictive of the efficacy ofcancer chemotherapeutic compounds.

EXAMPLES Example 1 Preparation of γ-glutamate-masked amoebapore analog

The twenty-five amino acid, C-terminal amidated, amoebapore cytolyticpeptide was synthesized by standard solid phase peptide synthesis,except that the ε amino group of the C-terminal lysine was blocked witha different blocking group from that used to block the ε-amino groups ofthe other two lysine residues in the peptide, so that the terminal εblock could be selectively removed. After selective removal of theblocking group from C-terminal lysine ε amino group, this amino groupwas linked to the γ carboxyl group of a first glutamate residue withblocked α amino and carboxyl groups by solution phase synthesis. Thisresults in the addition of a γ glutamate linked side-chain glutamic acidresidue.

The α amino group of this first glutamic acid residue was de-protectedand a second glutamate residue was then linked to the de-protected αamino group via a γ glutamate linkage between the γ carboxyl group ofthe second glutamic acid residue and the α amino group of the first γglutamate linked side-chain glutamic acid residue. The second γglutamate linked side-chain glutamic acid residue was also added bysolution phase chemistry. If desired, further γ glutamate linkedside-chain glutamic acid residues may be added in the same fashion.

Below is shown a diagram of the initial cytolytic peptide and theprocytolytic peptide synthesized by the addition of the two γ glutamatelinked side-chain glutamic acid residue to the ε amino group of theC-terminal lysine.

Cytolytic Peptide: (SEQ ID NO: 17)

N-Gly-Phe-Ile-Ala-Thr-Leu-Cys-Thr-Lys-Val-Leu-Asp-Phe-Gly-Ile-Asp-Lys-Ile-Gln-Leu-Ile-Glu-Asp-Lys-CONH₂.

Example 2 Assay for the Cytolytic Activity of the Pore-forming Toxins

For cell lysis assay, 10⁶ LNCaP prostate tumor cells were cultured in48-well plate in 200 μl culture medium containing differentconcentrations of the peptide. The culture medium for LNCaP cell wasRPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/Lsodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodiumpyruvate, 90%; fetal bovine serum 10%. The cells were treated withpeptide in a CO₂ incubator for one hour.

As seen in FIG. 1, human prostate cancer cells (LNCaP) are completelylysed after 15 minutes in 50 μM amoebapore helix 3-γ-Glu-γ-Glu, whilemouse L-cells remain unaffected after 24 hours under the sameconditions.

In order to obtain a more quantitative result, the assay was repeatedand decrease in ATP levels was used as a surrogate marker for liveversus dead cells. ATP plays a central role in the energy status of thecell and the regulation of enzymatic activity. The intracellular ATPlevel is strictly regulated. The assay of ATP has been used as a rapidand convenient measure of viable cell numbers. ATP level can beenzymatically determined with firefly luciferase, which specificallycatalyzes the hydrolysis of ATP. The amount of light generated by thisenzymatic reaction is directly related to the amount of ATP in theoriginal sample.

For this purpose, the ATP Bioluminescence Assay Kit CLS II fromBoehringer-Mannheim (Cat. No. 1699695) was employed. Briefly, 10⁶ cellsin 200 μl of medium containing the peptide, as above, were incubated at37° C. in a CO₂ incubator for 60 min. The cells were then lysed with 10μl of 10% Triton X-100. Each lysate was transferred into a tubecontaining 155 μl of distilled water and 25 μl of stock luciferase andchemiluminescence was measured in a luminometer as directed by themanufacturer. As can been seen from FIG. 3, ATP is dramatically depletedin the presence of either 50 or 100 μM peptide, as compared to thecontrols.

Example 3 Specificity for Additional Tumor Types and Lack of Toxicity

This example demonstrates that the inventive γ-glutamate-maskedcytolytic peptides have specificity for cancer cells other than thoseexpressing PSMA. This experiment, utilized a melittin analog having A[ε-γ]-Glu-[α-γ]-Glu at each of lysines 21 and 23:NH₂-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys([ε-γ]-Glu-[α-γ]-Glu)-Arg-Lys([ε-γ]-Glu-[α-γ]-Glu)-Arg-Gln-Gln-COOH.(SEQ ID NO: 19) Two prostate tumors (PNCap and DU0145), two ovariantumors (HeLa and SK-OV-3), one lung tumor (LLC 1) and one melanoma (B16) were tested. Cultured cells were treated with 1, 10, 50 or 100 μMpeptide. Results, depicted in FIG. 4, show strong lytic activity againstall tumors.

The same analog was used to treat a PSMA-expressing prostate cells(LNCaP-FCG), ovarian cancer cells (HeLa) or prostate cells notexpressing PSMA (PC3). While 25 and 50 mM concentrations were highlytoxic to the PSMA-expressing prostate cells and the ovarian cancercells, the non-PSMA-expressing prostate cells were relativelyunaffected. See FIG. 5.

To assess the toxicity of this analog, mice were treated with escalatingdoses of analog, either subcutaneously or intravenously. At 50, 100, 150and 200 mM concentrations (50-200-fold higher concentrations needed tokill a mouse using the unmasked melittin peptide) mice remained healthy,apparently unaffected, for weeks following treatment. These dataindicate that the subject peptides should be safe and effective intreating a variety of tumors.

Example 4 Treatment of Prostate Tumors with Melittin-Gamma Glutamate InVivo.

Four LNCaP-FCG tumors in nude mice were treated withmelittin-gamma-glutamate. Treatment was initiated when the tumordiameter was approximately 2.0 mm in diameter. 33 mg/kgmelittin-gamma-glutamate was administered every other day for fiveweeks. Results, depicted in FIG. 6, indicate that tumor volume remainedlow (less than 500 cu. mm) in all treated mice for the entire treatmenttime. The tumor volume continuously increased in the untreated controlanimal to 2000 cu. mm by day 15.

Day Volume 1 (mm cu) Volume 2 (mm cu) Volume 3 (mm cu) Volume 4 (mm cu)Untreated Control 1 — 270 — — 150 3 — 250 — — 500 5 14 175 — — 950 8 7.5269.5 125 — 1200 10 14 231 110 — 1500 12 15.75 486 342 — 1850 15 18 437150 20.25 2024 18 20 378 175 12.25 — 20 22.5 293.25 170.625 12.25 — 2340 288 175 9 — 25 60 280 180 9 — 27 60.5 280.5 135 7.5 — 30 28 165 847.5 — 32 25 140 84 6 —

Example 5 Preparation of Procytotoxins Inactivated by an Inhibitor

This example demonstrates that lytic peptides can be inactivated by theattachment of an inactivator that sterically hinders lytic peptideinsertion and pore conformation, rendering the peptide non-toxic.

As an example, melittin may be inactivated by attaching a phagefilament, other peptide, or a biotin-streptavidin microbead, accordingto the following:Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Gly-Ala-Ile*Gly-Gln-Pro]-(X)(SEQ ID NOS 1 & 2, respectively) wherein*denotes an MMP cleavage siteand (X) is an inactivator. MMP cleavage will yield a melittin peptidewith three additional amino acids on the C-terminus (Gly-Ala-Ile), whichshould not interfere with pore formation.

Other cleavage sites can also be used. If a prostate specific model isdesired, a prostate specific antigen (PSA) cleavage site can be employedby replacing the MMP site for Ser-Ser-Phe(or Tyr)-Tyr, or any of theother PSA cleavage sites. PSA is an endopeptidase and sequences for PSAcleavage have already been identified (Coombs et al, Chem Biol, 5(9):475-88, 1988).

The procytotoxin may further comprise a targeting sequence, such asBiotin-Gly-Gly-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ ID NO: 20)(RGD-4C) α_(v)β₃ integrin targeting peptide or biotin-anti-fibronectinED-B antibody.

1. A procytotoxin comprising at least one lysine residue bound via apeptide bond to at least one amino acid via the ε-amino group of saidlysine residue and further comprising a cytotoxic peptide bound to aninactivator via a peptide bond, wherein said peptide is a pore-formingcytolytic peptide that comprises an amphipathic alpha-helical structure,and wherein said peptide bond is susceptible to cleavage by PSMA.
 2. Theprocytotoxin of claim 1, further comprising a targeting molecule.
 3. Theprocytotoxin of claim 2, wherein said targeting molecule is selectedfrom the group consisting of a molecule that targets the neo-vasculatureand an antibody.
 4. The procytotoxin of claim 3, wherein said targetingmolecule is an RGD targeting sequence.
 5. The procytotoxin of claim 1,wherein said cytolytic peptide is selected from the group consisting ofAe I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore,amoebapore homolog from Entamoeba dispar, brevinin- 1 E, brevinin-2E,barbatolysin, cytolysin of Enterococcus faecalis, delta hemolysin,diphtheria toxin, E1 Tor cytolysin of Vibrio cholerae, equinatoxin,enterotoxin of Aeromonas hydrophila, esculentin, granulysin, haemolysinof Vibrio parahaemolyticus, intermedilysin of Streptococcus intermedius,the lentivirus lytic peptide, leukotoxin of Actinobacillusactinomycetemcomitans, magainin, melittin, membrane-associatedlymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1,neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphinfragment 4, NK-lysin, paradaxin, perform perfringolysin O, theta-toxin,of Clostridium perfringens, phallolysin, phallotoxin, streptolysin,D,L-α-amino acid cyclic peptides.
 6. The procytotoxin of claim 1,wherein said cytolytic peptide is a melittin.
 7. The procytotoxin ofclaim 6, wherein said cytolytic peptide comprises the followingstructure:Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Gly-Ala-Ile-Gly-Gln-Pro](residues1-32 of SEQ ID NOS 1 or 2).
 8. The procytotoxin of claim 7, furthercomprising a targeting molecule.
 9. A pharmaceutical composition,comprising one or more procytotoxins of claim 7 and a pharmaceuticallysuitable carrier or excipient.
 10. The procytotoxin of claim 6, whereinsaid cytolytic peptide comprises the following structure:Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Ser-Ser-Phe(orTyr)-Tyr-Ser-Gly(or Ser)] (residues 1-32 of SEQ ID NOS 3 or 4).
 11. Theprocytotoxin of claim 10, further comprising a targeting molecule.
 12. Apharmaceutical composition, comprising one or more procytotoxins ofclaim 10 and a pharmaceutically suitable carrier or excipient.
 13. Apharmaceutical composition, comprising one or more procytotoxins ofclaim 1 and a pharmaceutically suitable carrier or excipient.
 14. Theprocytotoxin of claim 1, wherein said inactivator is selected from thegroup consisting of a microbead, an amino acid, a peptide, phage and aphage filament.
 15. A method for selectively destroying a target cell,comprising contacting a target cell expressing PSMA with a procytotoxin,which comprises a cytotoxic peptide bound via a peptide bond to aninactivator, wherein said peptide is a pore-forming cytolytic peptidethat comprises an amphipathic alpha-helical structure, wherein saidprocytotoxin further comprises at least one lysine residue bound via apeptide bond to at least one amino acid via the ε-amino group of saidlysine residue, and wherein said peptide bond is susceptible to cleavageby PSMA.
 16. The method of claim 15, wherein said procytotoxin furthercomprises a targeting molecule.
 17. The method of claim 16, wherein saidtargeting molecule is selected from the group consisting of a moleculethat targets the neo-vasculature and an antibody.
 18. The method ofclaim 17, wherein said targeting molecule is an RGD targeting sequence.19. The method of claim 15, wherein said cytolytic peptide is selectedfrom the group consisting of Ae I, cytolysin of sea anemone, aerolysin,amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar,brevinin- 1E, brevinin-2E, barbatolysin, cytolysin of Enterococcusfaecalis, delta hemolysin, diphtheria toxin, E1 Tor cytolysin of Vibriocholerae, equinatoxin, enterotoxin of Aeromonas hydrophila, esculentin,granulysin, haemolysin of Vibrio parahaemolyticus, intermedilysin ofStreptococcus intermedius, the lentivirus lytic peptide, leukotoxin ofActinobacillus actinomycetemcomitans, magainin, melittin,membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin,neokyotorphin fragment 1, neokyotorphin fragment 2, neokyotorphinfragment 3, neokyotorphin fragment 4, NK-lysin, paradaxin, perforin,perfringolysin O, theta-toxin, of Clostridium perfringens, phallolysin,phallotoxin, streptolysin, D,L- α-amino acid cyclic peptides.
 20. Themethod of claim 15, wherein said cytolytic peptide is a melittin. 21.The method of claim 20, wherein said cytolytic peptide comprises thefollowing structure:Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Gly-Ala-Ile-Gly-Gln-Pro](residues 1-32 of SEQ ID Nos 1 or 2).
 22. The method of claim 21,further comprising a targeting molecule.
 23. The method of claim 20,wherein said cytolytic peptide comprises the following structure:Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Ser-Ser-Phe(orTyr)-Tyr-Ser-Gly(or Ser)] (residues 1-32 of SEQ ID Nos 3 or 4).
 24. Themethod of claim 23, further comprising a targeting molecule.
 25. Themethod of claim 15 wherein the target cell is a prostate cancer cell.26. The method of claim 15, wherein said inactivator is selected fromthe group consisting of a microbead, an amino acid, a peptide, phage anda phage filament.
 27. A method of making a Procytotoxin, comprisingmodifying a cytotoxic peptide to include an inactivator, wherein saidcytoxic peptide is a pore-forming cytolytic peptide that comprises anamphipathic alpha-helical structure; wherein said procytotoxin furthercomprises at least one lysine residue bound via a peptide bond to atleast one amino acid via the ε-amino group of said lysine residue, andwherein the inactivator is cleaved from the procytotoxin by PSMA. 28.The method of claim 27, wherein said procytotoxin further comprises atargeting molecule.
 29. The method of claim 28, wherein said targetingmolecule is selected from the group consisting of a molecule thattargets the neo-vasculature and an antibody.
 30. The method of claim 29,wherein said targeting molecule is an RGD targeting sequence.
 31. Themethod of claim 27, wherein said cytolytic peptide is selected from thegroup consisting of Ae I, cytolysin of sea anemone, aerolysin, amatoxin,amoebapore, amoebapore homolog from Entamoeba dispar, brevinin-1E,brevinin-2E, barbatolysin, cytolysin of Enterococcus faecalis, deltahemolysin, diphtheria toxin, El Tor cytolysin of Vibrio cholerae,equinatoxin, enterotoxin of Aeromonas hydrophila, esculentin,granulysin, haemolysin of Vibrio parahaemolyticus, intermedilysin ofStreptococcus intermedius, the lentivirus lytic peptide, leukotoxin ofActinobacillus actinomycetemcomitans, magainin, melittin,membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin,neokyotorphin fragment 1, neokyotorphin fragment 2, neokyotorphinfragment 3, neokyotorphin fragment 4, NK-lysin, paradaxin, performperfringolysin O, theta-toxin, of Clostridium perfringens, phallolysin,phallotoxin, streptolysin, D,L-α-amino acid cyclic peptides.
 32. Themethod of claim 27, wherein said cytolytic peptide is a melittin. 33.The method of claim 27, wherein said pore-forming cytolytic peptidecomprises the following structure:Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Gly-Ala-Ile-Gly-Gln-Pro](residues1-32 of SEQ IDNOS 1 or 2).
 34. The method of claim 33, furthercomprising adding a targeting molecule to said procytotoxin.
 35. Themethod of claim 34 wherein said targeting molecule is selected from thegroup consisting of a molecule that targets neovascular and an antibody.36. The method of claim 32, wherein said cytolytic peptide comprises thefollowing structure:Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Ser-Ser-Phe(orTyr)-Tyr-Ser-Gly (or Ser)] (residues 1-32 of SEQ ID NOS 3 or 4).
 37. Themethod of claim 27, wherein said inactivator is selected from the groupconsisting of a microbead, an amino acid, a peptide, phage and a phagefilament.
 38. A method of treating a cancer patient comprisingadministering a therapeutically effective amount of the pharmaceuticalcomposition of claim 13 to a patient having prostate cancer.